Exosomes comprising therapeutic polypeptides

ABSTRACT

The present invention pertains to an inventive release mechanism for extracellular vesicle (EV)-mediated intracellular and intramembrane delivery of therapeutic polypeptides. More specifically, the invention relates to EVs comprising polypeptide constructs which comprise a therapeutic polypeptide releasably attached to an exosomal polypeptide. Furthermore, the present invention pertains to manufacturing methods, pharmaceutical compositions, medical uses and applications, and various other embodiments related to the inventive EVs.

RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 16/304,580,filed on Nov. 26, 2018, which is a U.S. National Phase application,filed under 35 U.S.C. § 371, of International Application No.PCT/GB2017/051479, filed on May 25, 2017, which claims priority to andthe benefit of GB 1609216.5, filed on May 25, 2016, the entire contentsof each of which are incorporated herein by reference.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the .xml file named “EVOX-002_D01US_SeqList_ST26”, whichwas created on Nov. 15, 2022 and is 2,761 bytes in size, are herebyincorporated by reference in their entirety.

TECHNICAL FIELD

The present invention pertains to extracellular vesicle (EV)therapeutics, wherein the EVs comprise at least one polypeptide ofinterest (Pol).

BACKGROUND TO THE INVENTION

The exquisite specificity between an antibody and its antigen, or forthat matter between any type of protein-based biopharmaceutical and itstarget, is an ideal basis for therapeutic intervention. However, thetherapeutic use of antibodies and protein biologics is limited toextracellular targets because of the highly restricted access of largemolecular species to the intracellular environment. Various vehicles areunder investigation for the delivery of therapeutic polypeptides to thecell interior and recent research has shown the utility of e.g.cell-penetrating peptides (reviewed for instance by Dinca et al., Int JMol Sci, 2016) and bispecific antibodies which target existing transportpathways (Yu et al., Sci Trans Med, 2014).

A completely different approach was taken in the seminal patentapplication WO2013/084000, which discloses the use of exosomes forintracellular delivery of biotherapeutics. More specifically,WO2013/084000 discloses how polypeptide-based therapeutics may be loadedinto exosomes both via exogenous and endogenous loading techniques.Exogenous loading of exosomes may be carried out using electroporationor transfection of the polypeptide of interest into exosomespost-isolation from the parental cell, whereas endogenous loading isbased on transfection of the parental cell with a construct encoding thepolypeptide of interest, followed by overexpression of the construct andharvesting of exosomes comprising the biotherapeutic polypeptide.

Another groundbreaking patent application (WO2014/168548) disclosestherapeutic delivery vesicles, such as exosomes, having attached totheir membrane a polypeptide construct comprising at least one carrierpolypeptide fused to at least one therapeutic polypeptide, which ispresent at least partially on the outside of the vesicle, so that it isdisplayed to the extravesicular environment. Other patent applicationshave attempted to use exosomes for the delivery of protein biologics,such as, in the case of WO2015/138878, heparin-binding epidermal growthfactor (HB-EGF).

However, successful intracellular delivery of bioactive proteinbiologics, especially antibodies/intrabodies and other polypeptidesintended to interact with a specific intracellular target, oftennecessitates that the therapeutic polypeptide of interest is deliveredwith high efficacy in its free and unconjugated form. The exogenousloading of exosomes post isolation is often a cumbersome and ineffectivestrategy for loading of polypeptides, and similarly the endogenousloading strategies of the prior art implies that intraluminal EV loadingis either inefficient or that the polypeptide of interest (Pol) is notpresent in its bioactive unconjugated form. Recently, WO2016/178532described an optogenetic method for creating protein-carrying EVs,wherein two complex dimeric optogenetic constructs are introduced into aparental cell. Upon exogenously applied light exposure, two differentoptogenetic proteins associate and upon ceased light exposure theproteins dissociate. This method requires extended long-term exposure ofbiological material to a light source in order to transport the proteininto exosomes and subsequently to release it from its exosomaltransporter. As a result, the method suffers from problems withscalability, potential toxicity issues, and is also highly cumbersome tocarry out, in part due to the long-term exogenous application of light.Furthermore, the dimeric construct means that there is a need formultiple vectors and that the risk of protein misfolding, toxic proteinaggregation, and translational errors increases substantially, inaddition to the risk of imperfect association and dissociation betweenthe proteins.

SUMMARY OF THE INVENTION

It is hence an object of the present invention to overcome theabove-identified problems associated with the delivery of proteinbiologics into a target cell, and to satisfy the existing needs withinthe art, for instance to enable reaching intracellular targets withantibodies or intrabodies, enzymes for enzyme replacement therapy, orgene editing enzymes, or any other type of polypeptide that is intendedto have a bioactive effect in a target cell or target tissue. Thepresent invention achieves this by utilizing extracellular vesicle(EV)-based therapeutics, which are based on exosomes or any other typeof EVs comprising at least one polypeptide/protein of interest (Pol),wherein the Pol is released from an endogenously activatablepolypeptide-based release system and subsequently delivered into atarget cell, e.g. specifically into a suitable cellular compartment orinto an organelle of a target cell. The release system of the presentinvention is based on a fusion protein between an exosomal polypeptideand a domain that enables endogenously activatable releasable attachmentof the Pol, meaning that the Pol may be released through an endogenousactivation trigger into one of more of e.g. the lumen of an EV, into themembrane of an EV, or into any compartment or organelle of a target cellor target tissue. Importantly, endogenous activation of thepolypeptide-based release domain means that the method is highlyscalable, contains no extra steps during which biological material isexposed for extended time periods to potentially toxic exogenous stimulior conditions, and is transferable across therapeutics platforms.Without wishing to be bound by any theory, it is surmised that theendogenous activation may be a result of e.g. a drop in pH, competitionfor binding partners, and generally any change of cell biologicalconditions which may be conducive to triggering release of the Polthrough activation of the polypeptide-based release domain.

This highly sophisticated approach to the delivery of unconjugatedtherapeutic polypeptides of interest into the lumen or the membrane ofan EV enables extremely efficient delivery of bioactive polypeptides oftherapeutic interest into the intracellular environment and/or to anytype of cellular membrane structure (such as the plasma membrane, themembrane of an organelle such as the nucleus, a lysosome or theendoplasmic reticulum (ER) or any other type of membrane compartment) ofa target cell. The present invention may thus be applied to any type ofintracellular and/or intramembrane delivery of polypeptides, forinstance for the introduction or replacement of any type of protein orpeptide. A non-limiting example may be an enzyme that is absent orinactive in diseases involving e.g. a genetic abnormality, such aslysosomal storage diseases, or any type of intracellular or integralmembrane protein that needs to be replaced or be present in a higherconcentration in a target. As a further non-limiting example, thepresent invention is highly useful in the treatment of cancer, whereinthe EVs of the present invention may be utilized to introduce into theintracellular environment a tumor suppressor protein (or a variant orderivative thereof) such as p53 or p21 or any type of polypeptide (suchas an intrabody or an antibody) that binds to and inhibits a tumorigenicpathway. Yet other non-limiting examples include to delivertranscription factors or components of signaling pathways for modulatinginflammatory responses or induce tissue repair, or to deliverRNA-binding proteins which may carry with them single or double RNAstrands which may themselves confer therapeutic activity.

The present invention thus pertains inter alia to EVs comprising atleast one Pol, wherein the at least one Pol is attached to an exosomalpolypeptide via an endogenously activatable release system. For clarity,the present invention thus relates to both EVs comprising a Pol whichhas been released into the lumen of an EV and/or into the membrane of anEV by a release system, and EVs comprising a Pol which is still attachedin a releasable manner to an endogenously activatable polypeptide-basedrelease system. The modularity of the polypeptide constructs of thepresent invention (which comprises a polypeptide of interest, apolypeptide-based endogenously activatable release domain, and anexosomal polypeptide) enables a highly controllable endogenousproduction of EVs for the delivery of unconjugated Pols (asabove-mentioned either as a soluble Pol or as a membrane-associated Pol,wherein the Pol may face either the external environment or the internalenvironment or both in a transmembrane fashion). This is in completecontrast to the prior art, which merely describes endogenous loading ofPols which are either permanently conjugated to exosomal proteins orPols loaded into EVs without the aid of any exosomal polypeptide, orPols which are transported and released into EVs as a result of extendedlong-term exposure to an exogenous light source, which means that theloading and the production processes are extremely inefficient,cumbersome, un-scalable and/or potentially toxic in comparison toloading and EV production as per the present invention.

Furthermore, the present invention pertains to several novel methods forloading and production of EVs comprising Pols, cells comprisingpolynucleotide and/or polypeptide constructs enabling such production,and inventive polynucleotide and/or polypeptide constructs as such. Morein detail, the present invention relates to the use of cis-cleavingpolypeptides (i.e. polypeptides or peptides comprising specificsequences of amino acids that trigger release (which can take place by avariety of mechanisms, e.g. splicing or cleavage) of desired parts ofthe peptide to in turn release the Pol) and nuclear localization signal(NLS)-binding polypeptides (NLSBPs) (e.g. an importin alpha polypeptide)for EV-mediated delivery of intraluminal and/or membrane-associatedunconjugated polypeptides of interest (Pols). In one separateembodiment, the present inventors have contemplated the use of a simplemonomeric polypeptide-based release system which can be triggered by avery rapid light boost (as opposed to extended light exposure-basedtransport and release systems such as in WO2016/178532).

The present invention further pertains to methods for intracellulardelivery of unconjugated Pols, wherein such methods comprise the stepsof contacting a target cell with an EV comprising (i) a Pol releasablyattached to an exosomal polypeptide via endogenously triggered releasedomain(s) or (ii) a Pol released via endogenous activation from anexosomal polypeptide (normally inside the lumen or the membrane of theEV). The EV may typically enter the target cell and deliver itspolypeptide cargo, resulting in highly efficient intracellular orintramembrane delivery of the therapeutic polypeptide. The EVs and themethods for their production and for intracellular delivery thus haveextensive medical potential, for instance in the prophylaxis and/ortreatment of a large number of diseases and ailments, notably withinoncology, inflammation and autoimmunity, neuroinflammatory andneurodegenerative disorders, genetic diseases, lysosomal storagedisorders, organ injuries and failure, muscular dystrophies such as DMD,cardiovascular and metabolic disorders, kidney and liver diseases suchas non-alcoholic steatohepatitis (NASH), etc.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic illustration of a polypeptide constructcomprising a cis-cleaving release system (such as an intein) for releaseof a polypeptide of interest (POI).

FIG. 2 shows a general illustration of a polypeptide constructcomprising a monomeric short-term light-induced cleavage release systemfor release of a Pol. A short-term light boost leads to cleavage of therelease system and thereby release of the Pol, without any toxic effectsand without issues with scalability.

FIG. 3 shows the results of GBA-deficient cells treated with EVsenriched with intein-GBA enriched fusion proteins. Recipient cellsexperience a decrease in glucocerebroside levels following the deliveryof bioactive GBA. A similar experiment was carried out with thetransporter NPC1 in NPC1-deficient fibroblasts.

FIG. 4 depicts a polypeptide construct comprising a light-inducedcleavage release system based on the GFP-like Dendra protein forreleasing a Pol. Dendra is a monomeric release system that can beactivated by a short-term boost of light.

FIG. 5 shows the results of a non-homologous end-joining (NHEJ) assay.HEK293T-red cells containing a reporter system were transfected withexosomes comprising guide RNA (gRNA) and a polypeptide constructcomprising CD81 as the exosomal polypeptide, Kaede as the monomericlight-induced cleavage-based release system, and Cas9 as the Pol.Exosomes where obtained from a cell culture that either was or was notexposed to a short-term boost of blue light during the exosomeproduction process. Only exosomes obtained from cells exposed tolight—which induced cleavage of the Kaede and thereby release ofCas9—showed an increase in the percentage of positive cells.

FIG. 6 shows the results of a High Resolution Melting (HRM) analysis ofcells treated with EVs harvested from cells exposed to light. EVscomprising the same polypeptide construct as in FIG. 5 inducedCas9-mediated mutations in the AAVS locus as a result of efficientintracellular delivery of bioactive Cas9 and gRNA.

FIG. 7 shows the effects in the NHEJ assay of EVs comprising apolypeptide construct comprising Cas9 fused to CD63 through themonomeric light-induced release polypeptide Dendra2, which rendersfunctional Cas9 in EVs after UV/blue light irradiation.

FIG. 8 shows HRM results of cells treated with EVs harvested from cellsexposed to UV/blue light. EVs comprising the same polypeptide constructas in FIG. 7 induced Cas9-mediated mutations in the AAVS locus as aresult of efficient intracellular delivery of bioactive Cas9 and gRNA.

FIG. 9 shows robust down regulation of NfKB response achieved by EVsloaded with a WASP-targeted single-chain variable fragment(scFv)-KikGR-CD63 polypeptide construct, exposed to UV-light to releasethe scFv inside the target bone marrow—derived macrophage cell.

FIG. 10 shows the results of Jurkat cells stimulated to express theIL2-receptor and at the same time treated with EVs loaded with a scFvtowards the IL2R-alpha subunit. Only the positive control and the EVsloaded with scFv-Dendra2-CD63 (and exposed to UV-light) induced a downregulation of the IL2R on the cell surface of the Jurkat cells,according to FACS analysis.

FIG. 11 shows the results of the erbB-2-positive ovarian carcinoma cellline SKOV3 treated with EVs loaded with a scFv targeted towards theoncoprotein erb-2. Cell death was assayed at 48 hours after treatment.Only EVs loaded with scFv-Dendra2-CD63 (and thereafter exposed toUV-light) induced cell death in comparable levels to the positivecontrol.

FIG. 12 shows the results of loading NRF2 transcription factor to EVsusing the NLSBP-NLS-based release system and the associated effects ininducing target gene HMOX1 expression in recipient cells. An NLSBP(KPNA1 a.k.a. importin α5) was fused to exosomal protein CD63 andco-expressed in EV source cells (various types of immune cells weretested with good results) with NRF2. Co-expression leads tosignificantly enhanced EV sorting of NRF2, as estimated by Westernblotting as compared to expression of NRF2 alone. Delivery of NRF2loaded EVs using this strategy leads to induction of target geneexpression in EV recipient cells.

FIG. 13 shows a similar experiment as in FIG. 5 , but here with acis-cleaving intein (comprising the amino acid sequenceVal-Val-Val-His-Asn (SEQ ID NO: 1)) as a release system fused to CD63,CD81 (data not shown), and syntenin (data not shown) and to Cas9. As canbe seen from FIG. 13 , only the unmutated intein induced relevant levelsof NHEJ.

FIG. 14 shows an illustration of a Western blot analysis of Crerecombinase enrichment within exosomes using an intein-based polypeptidereleasable system. Lanes 1-3 shows exosomes and lanes 5-7 theirrespective whole cell lysate. Lane 1 and 5—Soluble NLSCre; Lanes 2&6CD63-intein-Cre; Lanes 3&7 CD63-intein-NLSCre; 4 protein ladder; Alixloading control; Cre recombinase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to EVs comprising a polypeptideconstruct, which in turn comprises (i) at least one Pol, (ii) at leastone exosomal polypeptide, and (iii) at least one polypeptide-basedrelease system, wherein the at least one Pol is releasably attached tothe at least one exosomal polypeptide with the aid of the releasesystem, wherein the release system is endogenously activatable and therelease of the Pol is thus triggered automatically without beingdependent on any exogenous stimuli. Further, the present invention alsorelates to EVs comprising at least one Pol which has been released fromthe at least one exosomal polypeptide with the aid of the release systemin the EV, either essentially in the lumen of the EV or in association(e.g. into) with the EV membrane. Furthermore, the invention relates tovarious related aspects as will be described in greater detail below,for instance polynucleotide and polypeptide constructs and cellscomprising such constructs, production methods and methods forintracellular delivery of polypeptides/proteins of interest in vitro andin vivo, as well as medical applications of such EVs and pharmaceuticalcompositions containing such EVs.

For convenience and clarity, certain terms employed herein are collectedand described below. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs.

Where features, aspects, embodiments, or alternatives of the presentinvention are described in terms of Markush groups, a person skilled inthe art will recognize that the invention is also thereby described interms of any individual member or subgroup of members of the Markushgroup. The person skilled in the art will further recognize that theinvention is also thereby described in terms of any combination ofindividual members or subgroups of members of Markush groups.Additionally, it should be noted that embodiments and features describedin connection with one of the aspects and/or embodiments of the presentinvention also apply mutatis mutandis to all the other aspects and/orembodiments of the invention. For example, the various at least onepolypeptides of interest (Pol) described in connection with the EVs isto be understood to be disclosed and relevant also in the context of thepolypeptide constructs or in the context of the pharmaceuticalcompositions comprising EVs, or as expression products of thepolynucleotide constructs as per the present invention. Furthermore,certain embodiments described in connection with certain aspects, forinstance the administration routes of the EVs, as described in relationto aspects pertaining to treating certain medical indications, maynaturally also be relevant in connection with other aspects and/orembodiment such as aspects/embodiments pertaining to the pharmaceuticalcompositions or the intracellular delivery methods of the presentinvention. As a general remark, the polypeptides of interest (Pol), theexosomal polypeptides, the endogenously activatable release systems, andthe targeting moieties, the cell sources, and all other aspects,embodiments, and alternatives in accordance with the present inventionmay be freely combined in any and all possible combinations withoutdeviating from the scope and the gist of the invention. Furthermore, anypolypeptide or polynucleotide or any polypeptide or polynucleotidesequences (amino acid sequences or nucleotide sequences, respectively)of the present invention may deviate considerably from the originalpolypeptides, polynucleotides and sequences as long as any givenmolecule retains the ability to carry out the technical effectassociated therewith. As long as their biological properties areretained the polypeptide and/or polynucleotide sequences according tothe present application may deviate with as much as 50% (calculatedusing for instance BLAST or ClustalW) as compared to the nativesequence, although a sequence identity that is as high as possible ispreferable. The combination (fusion) of e.g. at least one polypeptide ofinterest and at least one peptide/polypeptide-based release system andat least one exosomal polypeptide implies that certain segments of therespective polypeptides may be replaced and/or modified, meaning thatthe deviation from the native sequence may be considerable as long asthe key properties are conserved. Similar reasoning thus naturallyapplies to the polynucleotide sequences encoding for such polypeptides.

The terms “extracellular vesicle” or “EV” or “exosome” shall beunderstood to relate to any type of vesicle that is, for instance,obtainable from a cell, for instance a microvesicle (e.g. any vesicleshed from the plasma membrane of a cell), an exosome (e.g. any vesiclederived from the endo-lysosomal pathway), an apoptotic body (e.g.obtainable from apoptotic cells), a microparticle (which may be derivedfrom e.g. platelets), an ectosome (derivable from e.g. neutrophils andmonocytes in serum), prostatosome (e.g. obtainable from prostate cancercells), or a cardiosome (e.g. derivable from cardiac cells), etc.Furthermore, the said terms shall also be understood to relate tolipoprotein particles, such as LDL, VLDL, HDL and chylomicrons, as wellas extracellular vesicle mimics, cellular membrane vesicles obtainedthrough membrane extrusion or other techniques, etc.

Essentially, the present invention may relate to any type of lipid-basedstructure (with vesicular morphology or with any other type of suitablemorphology) that can act as a delivery or transport vehicle for thepolypeptide of interest (Pol) and polypeptide constructs containing suchPols. It will be clear to the skilled artisan that when describingmedical and scientific uses and applications of the EVs, the presentinvention normally relates to a plurality of EVs, i.e. a population ofEVs which may comprise thousands, millions, billions, trillions or evenquadrillions or quintillions (e.g. 10³-10¹⁸) of EVs, or even greaterpopulations of EVs (>10¹⁸ of EV particles). In the same vein, the term“population”, which may e.g. relate to an EV comprising a certain typeof Pol and/or a certain type of polypeptide construct comprising a Pol,shall be understood to encompass a plurality of entities (typicallycounted as particles) constituting such a population. In other words,individual EVs when present in a plurality constitute an EV population.Thus, naturally, the present invention pertains both to individual EVscomprising various Pols and populations comprising EVs which in turncomprise various Pols, as will be clear to the skilled person.

The terms “exosomal polypeptide” and “exosomal protein” and “EVpolypeptide” and “EV protein” are used interchangeably herein and shallbe understood to relate to any polypeptide that can be utilized totransport a polypeptide construct (which typically comprises, inaddition to the exosomal protein, at least one polypeptide of interestand at least one polypeptide-based release system) to a suitablevesicular structure, i.e. to a suitable EV. More specifically, the term“exosomal polypeptide” shall be understood as comprising any polypeptidethat enables transporting, trafficking or shuttling of a polypeptideconstruct (which as abovementioned typically comprises at least one Poland at least one polypeptide based release system, but which may alsoinclude a targeting peptide/polypeptide) to a vesicular structure, suchas an exosome. Examples of such exosomal polypeptides are for instanceCD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133,CD138, CD235a, ALIX, Syntenin-1, Syntenin-2, Lamp2b, TSPAN8, TSPAN14,CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1,DLL4, JAG1, JAG2, CD49d/ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c,CD18/ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, Fc receptors,interleukin receptors, immunoglobulins, MHC-I or MHC-II components, CD2,CD3 epsilon, CD3 zeta, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L,CD44, CD45, CD45RA, CD47, CD86, CD110, CD111, CD115, CD117, CD125,CD135, CD184, CD200, CD279, CD273, CD274, CD362, COL6A1, AGRN, EGFR,GAPDH, GLUR2, GLUR3, HLA-DM, HSPG2, L1CAM, LAMB1, LAMC1, LFA-1,LGALS3BP, Mac-1 alpha, Mac-1 beta, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD,TCRG, VTI1A, VTI1B, and any combinations thereof, but numerous otherpolypeptides capable of transporting a polypeptide construct to an EVare comprised within the scope of the present invention. The EV proteinsare typically of human origin and can be found in various publiclyavailable databases such as Uniprot, RCSB, etc.

The terms “polypeptide of interest”, “protein of interest”, “therapeuticpolypeptide of interest”, “Pol”, “biotherapeutic”, “biologic”, and“protein biologic” are used interchangeably herein and shall beunderstood to relate to any polypeptide that can be utilized fortherapeutic purposes through e.g. binding a target and/or in any otherway interacting with an interaction partner and/or replace a proteinand/or supplement or complement an existing intracellular protein,thereby exerting its therapeutic effect. Said terms may represent thefollowing non-limiting examples of therapeutic polypeptides of interest:antibodies, intrabodies, single chain variable fragments (scFv),affibodies, bi-och multispecific antibodies or binders, receptors,ligands, enzymes for e.g. enzyme replacement therapy or gene editing,tumor suppressors, viral or bacterial inhibitors, cell componentproteins, DNA and/or RNA binding proteins, DNA repair inhibitors,nucleases, proteinases, integrases, transcription factors, growthfactors, apoptosis inhibitors and inducers, toxins (for instancepseudomonas exotoxins), structural proteins, neurotrophic factors suchas NT3/4, brain-derived neurotrophic factor (BDNF) and nerve growthfactor (NGF) and its individual subunits such as the 2.5S beta subunit,ion channels, membrane transporters, proteostasis factors, proteinsinvolved in cellular signaling, translation- and transcription relatedproteins, nucleotide binding proteins, protein binding proteins, lipidbinding proteins, glycosaminoglycans (GAGs) and GAG-binding proteins,metabolic proteins, cellular stress regulating proteins, inflammationand immune system regulating proteins, mitochondrial proteins, and heatshock proteins, etc. In one preferred embodiment, the Pol is aCRISPR-associated (Cas) polypeptide with intact nuclease activity whichis associated with (i.e. carries with it) an RNA strand that enables theCas polypeptide to carry out its nuclease activity in a target cell oncedelivered by the EV. Alternatively, in another preferred embodiment, theCas polypeptide may be catalytically inactive, to enable targetedgenetic engineering. Yet another alternative may be any other type ofCRISPR effector such as the single RNA-guided endonuclease Cpf1. Theinclusion of Cpf1 as the Pol is a particular preferred embodiment of thepresent invention, as it cleaves target DNA via a staggereddouble-stranded break, Cpf1 may be obtained from species such asAcidaminococcus or Lachnospiraceae. In yet another exemplary embodiment,the Cas polypeptide may also be fused to a transcriptional activator(such as the P3330 core protein), to specifically induce geneexpression. Additional preferred embodiments include Pols selected fromthe group comprising enzymes for lysosomal storage disorders, forinstance glucocerebrosidases such as imiglucerase, alpha-galactosidase,alpha-L-iduronidase, iduronate-2-sulfatase and idursulfase,arylsulfatase, galsulfase, acid-alpha glucosidase, sphingomyelinase,galactocerebrosidase, galactosylceramidase, ceramidase,alpha-N-acetylgalactosaminidase, beta-galactosidase, lysosomal acidlipase, acid sphingomyelinase, NPC1, NPC2, heparan sulfamidase,N-acetylglucosaminidase, heparan-α-glucosaminide-N-acetyltransferase,N-acetylglucosamine 6-sulfatase, galactose-6-sulfate sulfatase,galactose-6-sulfate sulfatase, hyaluronidase, alpha-N-acetylneuraminidase, GlcNAc phosphotransferase, mucolipin1, palmitoyl-proteinthioesterase, tripeptidyl peptidase I, palmitoyl-protein thioesterase 1,tripeptidyl peptidase 1, battenin, linclin, alpha-D-mannosidase,beta-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,cystinosin, cathepsin K, sialin, LAMP2, and hexoaminidase. In otherpreferred embodiments, the Pol may be e.g. an intracellular protein thatmodifies inflammatory responses, for instance epigenetic proteins suchas methylases and bromodomains, or an intracellular protein thatmodifies muscle function, e.g. transcription factors such as MyoD orMyf5, proteins regulating muscle contractility e.g. myosin, actin,calcium/binding proteins such as troponin, or structural proteins suchas Dystrophin, utrophin, titin, nebulin, dystrophin-associated proteinssuch as dystrobrevin, syntrophin, syncoilin, desmin, sarcoglycan,dystroglycan, sarcospan, agrin, and/or fukutin. The Pols are typicallyproteins or peptides of human origin unless indicated otherwise by theirname, any other nomenclature, or as known to a person skilled in theart, and they can be found in various publicly available databases suchas Uniprot, RCSB, etc.

The term “released inside” as in the context of “released inside an EV”or “released inside a target cell” can be understood to mean release ofa polypeptide of interest (Pol) completely and/or partially inside theEV (or the target cell), i.e. that a Pol is released into the lumen ofan EV (or the target cell), into the membrane of an EV (or the targetcell) either completely or partially (e.g. into a transmembraneconfiguration) or onto the outside of the EV (or the target cell)membrane. Said term may also be understood to mean release of a Pol ontothe external side of the EV membrane (or the target cell). Furthermore,it may also include being released inside any biological system, e.g. aparticular tissue or a target organ.

The terms “endogenous activation”, “endogenous triggering” and variantsthereof (such as “endogenously activatable” or “endogenously triggered”)shall be understood to relate to activation, induction, and/ortriggering of release of the Pol by the release system without anyexogenous stimuli, i.e. the release of the Pol is triggered inside an EVor inside a cell by the mere action of the surrounding exosomal and/orcellular and/or biological environment (e.g. as a result of changes inpH, changes in other physiological parameters such as salinity,competition between binding partners, enzymatic activity e.g.proteolytic activity, etc.).

The terms “source cell” or “EV source cell” or “parental cell” or “cellsource” or “EV-producing cell” or any other similar terminology shall beunderstood to relate to any type of cell that is capable of producingEVs under suitable cell culturing conditions, for instance in suspensionculture or in adherent culture or any in other type of culturing system.The source cells per the present invention may be select from a widerange of cells, for instance mesenchymal stem or stromal cells(obtainable from e.g. bone marrow, adipose tissue, Wharton's jelly,perinatal tissue, tooth buds, umbilical cord blood, etc.), amnion cells,amnion epithelial cells, myeloid suppressor cells, immortalized celllines of which human embryonic kidney (HEK) cells represent onenon-limiting example, dendritic cells (DCs) or other immune system cellssuch as macrophages, monocytes, B- or T-cells, NK cells, neutrophils,eosinophils, mast cells or basophils, etc. Generally, EVs may be derivedfrom essentially any cell source, be it a primary cell source or animmortalized cell line. The EV source cells may be any embryonic, fetal,and adult somatic stem cell types, including induced pluripotent stemcells (iPSCs) and other stem cells derived by any method. When treatingneurological diseases, one may contemplate to utilize as source cellse.g. primary neurons, astrocytes, oligodendrocytes, microglia, andneural progenitor cells. The source cell may be either allogeneic,autologous, or even xenogeneic in nature to the patient to be treated,i.e. the cells may be from the patient himself or from an unrelated,matched or unmatched donor. In certain contexts, allogeneic cells may bepreferable from a medical standpoint, as they could provideimmuno-modulatory effects that may not be obtainable from autologouscells of a patient suffering from a certain indication. For instance, inthe context of treating peripheral or neurological inflammation,allogeneic MSCs may be preferable as EVs obtainable from such cells mayenable immuno-modulation via e.g. macrophage and/or neutrophilphenotypic switching (from pro-inflammatory M1 or N1 phenotypes toanti-inflammatory M2 or N2 phenotypes, respectively). Conversely, whenutilizing EVs for treating a solid or hematological malignancy, it maybe preferable to select immune cells such as DCs as the EV-producingcell source.

In a first aspect, the instant invention relates to an EV comprising atleast one polypeptide of interest (Pol), wherein the at least one Pol isreleasably attached to an exosomal polypeptide. The attachment of thePol to the exosomal polypeptide is releasable, in order to enableefficient, non-obstructed loading and endogenously triggered release ofthe therapeutic polypeptide of interest into the EV. The releasableattachment between the Pol and the exosomal polypeptide is a featuremediated by an inventive release system which enables endogenouslyactivatable release of the Pol inside the EV and/or subsequently insidea target cell or target tissue, to optimize loading and therapeuticactivity. The release system is a polypeptide-based system that may beselected from the group comprising various releasable polypeptideinteraction systems which may be activated or triggered without the needfor exogenous stimuli (i.e. the release systems are typically triggeredby endogenous activity within a cell or an EV, or essentially within anybiological system), for instance a cis-cleaving polypeptide-basedrelease system (e.g. based on inteins), a nuclear localization signal(NLS)— NLS binding protein (NLSBP)-based release system or releasesystems based on other protein domains. In one embodiment, a monomericlight-induced cleavage-based release system may be utilized, where onlya very short boost of light is utilized to start an endogenousproteolytic cleavage of a monomeric protein domain and release the Pol.

In a preferred embodiment, the present invention relates to an EVcomprising a polypeptide of interest which is releasably attached to anintein release system, and/or a polypeptide of interest (Pol) that hasbeen released from an intein release system inside the EV or inside atarget cell or target organ. A typical polypeptide construct thatemploys an intein release system can be described schematically asfollows (the below notation is not to be construed as illustrating any Cand/or N terminal direction, it is merely meant for illustrationpurposes):

-   -   Pol-Cis-cleaving polypeptide-Exosomal protein

Alternatively, the polypeptide construct may be designed as follows, toinclude a targeting moiety that will be displayed on the surface of theEV, to even further enhance its therapeutic potential by targeting atissue or cell type of interest:

-   -   Pol-Cis-cleaving polypeptide-Exosomal protein-Targeting Moiety

The cis-cleaving polypeptide-based release system may be either a fastor a slow cleaving release system. In certain instances, one may opt toutilize a fast-cleaving cis-cleaving release system (such as afast-cleaving cis-cleaving intein), whereas a slow-cleaving releasesystem may be advantageous in other settings. Generally, a slow-cleavingrelease system may be employed to allow longer time for loading of Polsinto EVs, whereas the fast-cleaving system may be preferable when EVsneed to be harvested quickly.

In a preferred embodiment, the cis-cleaving release system is based onan intein system, wherein the C-terminal portion of the intein maycomprise the amino acid sequences Val-Val-Val-His-Asn (SEQ ID NO: 1) orVal-Val-Val-His-Asn-Cys (SEQ ID NO: 2). Truncated or in other waysoptimized inteins, i.e. inteins where one or more amino acids have beenremoved or replaced to enhance functionality, may also be used for thepurposes of the present invention. Without wishing to be bound by anytheory it is surmised that truncation or increased-functionalitymutation may increase the pH responsiveness of the intein, which furtherincreases its utility in releasing bioactive Pols from EV-based deliverysystem as EV may be internalized into cells via endocytosis processes.However, more broadly, the cis-cleaving release system may be selectedfrom a group of cis-cleaving systems comprising various otherpolypeptide-based release systems, for instance Sortase A, N-terminusprotease, FrpC, and cysteine protease domains, or other suitablecis-cleaving release systems, and any combinations thereof. Thecis-cleaving release proteins may advantageously be attached tointraluminal exosomal polypeptide termini, to allow for release of thePol inside an EV or into an EV membrane, although other points ofattachment may also be employed, e.g. integral attachment points.

In one embodiment of the invention, the release of the Pol from theexosomal polypeptide which has guided the Pol to the EV may be achievedby light-induced cleavage. Unlike in the prior art, the presentinvention employs monomeric light-induced cleavage-based release systemswhich after a short light boost undergo endogenous activation and whichmay be selected from the group comprising monomeric proteins such asKaede, KikGR, EosFP, tdEosFP, mEos2, PSmOrange, and the GFP-like Dendraproteins Dendra and Dendra2. Unlike optogenetic dimerization systemssuch as CRY2-CIBN, monomeric proteins such as Dendra, Kaede, KikGR,EosFP, tdEosFP, mEos2, PSmOrange and Dendra2 have the advantages ofleaving only a small residual polypeptide domain on the Pol, which meansthat the bioactivity of the delivered Pol is not negatively affected.Furthermore, in contrast to optogenetic dimerization domains, thelight-induced cleavage-based release systems are considerably easier tocontrol, meaning that the loading of the Pol into the EV is highlyprecise. The light-induced cleavage-based released systems of thepresent invention thus enable highly controllable release of the Pol atdesired time points and at desired locations both in vitro and in vivo,simply by exposure to light of suitable wavelengths (in the case ofDendra, Kaede, KikGr and most other light-induced cleavage-based releaseproteins either UV or blue light, whereas in the case of PSmOrangelonger wavelengths in the red-orange spectrum). Importantly, acleavage-based light-induced release system is merely requiring a veryshort boost of light in order to effectuate an endogenous process ofcleavage, which means that potentially toxic effects of extended andcumbersome light exposure periods can be avoided. Polypeptide constructsbased on light-induced cleavage release systems may be describedschematically as follows (the below notation is not to be construed asillustrating any C and/or N terminal direction, it is merely meant forillustration purposes):

-   -   Pol-Dendra-Exosomal protein    -   Pol-Kaede-Exosomal protein

At a suitable time during the EV production process the polypeptideconstruct comprising the Pol, the light-induced cleavage system, and theexosomal polypeptide is exposed to a boost of light of a suitablewavelength, resulting in cleavage and release of the Pol. Uponshort-term exposure to UV or blue light, Dendra or other monomericUV/blue light-responsive light-induced cleavage release proteins, whichmay be inserted as a fusion between the Pol and the exosomalpolypeptide, are cleaved via an internal peptide backbone cleavage,liberating the Pol and the exosomal protein. Small polypeptide domainsmay remain on both the Pol and the exosomal polypeptide but the activityof the Pol is not hampered by the presence of these small residues.

In yet another embodiment of the present invention, the release systemmay be based on the interaction between a nuclear localization signal(NLS) binding polypeptide (NLSBP) and an NLS. The NLSBP-NLS releasesystem may comprise at least one Pol comprising at least one classicalor non-classical NLS. As a further alternative, NLS-like sequences(NLSLS) can be used that bind to the NLSBP but that do not triggernuclear import of the NLSLS Pol. The NLS may be a naturally occurringNLSLS or NLS (as would be the case with most Pols destined for thenucleus, e.g. transcription factors and nucleases) with or without anoverlapping RNA/DNA binding domain, or an NLS that is recombinantlyfused to a Pol which does not inherently comprise an NLS. The exosomalpolypeptide is in turn modified to comprise a suitable NLSBP (forinstance from the importin alpha or beta families e.g. the importinalpha KPNA1, or any other proteins involved in nuclear import),resulting in a releasable attachment between the NLS-containing Pol andthe NLSPB-containing exosomal polypeptide, upon endogenous activation ofthe NLS-NLSBP release system. The trigger of the release of the Pol istypically driven by competition between different NLS-NLSBP pairs, whichresults in release of the Pol. Polypeptide constructs based on theNLS-NLSBP release systems may be described schematically as follows (thebelow notation is not to be construed as illustrating any C and/or Nterminal direction, it is merely meant for illustration purposes):

-   -   Pol-NLS-NLSBP-Exosomal protein

As abovementioned, NLSBPs present in the target cell will (uponEV-mediated delivery of the NLS-containing Pol) out-compete theNLSBP-containing exosomal polypeptide, resulting in the Pol beingendogenously liberated and trafficked to the correct cellularcompartment. Naturally, the NLSBP-NLS-based release system is highlysuitable for polypeptides of interest that are meant to exert theirdesired activity in the nucleus and/or the nucleolus, however proteinsof interest destined to cytoplasm or other intracellular compartmentsare also compatible with the NLS-NLSBP release system, especially whenusing NLSLS instead of NLS. Non-limiting examples of NLSBP-NLS releasesystems may comprise the following: KPNA1-NRF2, KPNA6-STAT3,KPNB1-STAT3, KPNA2-and HSF1. NLSBPs may comprise importins from theimportin alpha and beta families, and other NLS-binding proteins,including KPNA1, KPNA2, KPNA3, KPNA4, KPNA5, KPNA6, KPNA7, KPNB1, IPO4,IPO5, IPO7, IPO8, IPO9, IPO11, IPO13, TPNO1, TNPO2, TNPO3, HIKESHI,SNUPN, HEATR3, and other RAN binding proteins. NLS-containing proteinsmay be selected from non-limiting examples such as transcriptionfactors, nucleases and other nuclear proteins such as CREB, C/EBP, bZIP,bHLH, MyoD, cMyc, SERBP, NF-1, Cys4, GATA-factors, OCT4, NANOG, KLF4,SOX2, HSF1, STAT3, SMAD3, p53, MEF2, SRF, NFkB, CAS9, Zinc fingernucleases, hnRNPA1, hnRNPA2, NUP153, RPL23A, RPS7, RPL5, RPL23A, H2A,H2B, H3 and H4 histones, TNRC6A, SRP19, SNAI1, PRKCI, HSP70, U1 snRNP,U2 snRNP, U4 snRNP, U5 snRNP, and U6 snRNP. Generally, non-limitingexamples of suitable Pols are for instance nucleases such as Cas andCas9 (which is an RNA-guided DNA endonuclease from Streptococcuspyogenes, among other bacteria); transcription-related proteins such asNF-κB and NRF2; DNA-binding proteins such as histones and polymerases;RNA-binding proteins such as hnRNPA1-2 and the MS2 coat protein whichmay be used to transport various types of RNAs; antibodies and/orintrabodies with nuclear targets, enzymes for enzyme replacementtherapies such as NPC1, NPC2 and GBA, etc. A preferred example of thepresent invention is fusing KPNA1 to CD63 and co-expressing with MyoD ina suitable EV source cell, such as an MSC or an amnion epithelial cell,thereby obtaining therapeutic EVs with strong applicability in treatinge.g. DMD.

As above-mentioned, the present invention relates to EV-basedtherapeutics comprising essentially any polypeptide of interest (Pol),typically for therapeutic or prophylactic purposes but potentially alsofor cosmetic uses. The Pol—or Pols in the cases where a plurality (i.emore than one) Pol are utilized—may be any suitable polypeptide, that isany molecule comprising a plurality of amino acids, i.e. a protein or apeptide. The Pol may be selected from anyone of the followingnon-limiting examples of therapeutic, prophylactic, or cosmeticpolypeptides: antibodies, intrabodies, single chain variable fragments(scFv), affibodies, bi-och multispecific antibodies or binders,receptors, ligands, enzymes such as enzymes lacking and/or defect inlysosomal storage diseases (LSDs), tumor suppressors such as p53, pVHL,APC, CD95, ST5, YPEL3, ST7, and ST14, viral or bacterial inhibitors,cell component proteins, DNA and/or RNA binding proteins, nucleases suchas Cas, Cas9, and Cpf1, proteinases, integrases, transcription factors,growth factors, apoptosis inhibitors and inducers, structural proteins,ion channels, membrane transporters, proteostasis factors, proteinsinvolved in cellular signaling, translation- and transcription relatedproteins, nucleotide binding proteins, protein binding proteins, lipidbinding proteins, glycosaminoglycans (GAGs) and GAG-binding proteins,metabolic proteins, cellular stress regulating proteins, inflammationand immune system regulating proteins, mitochondrial proteins, and heatshock proteins, etc. The fact that EVs enable reaching the intracellularmilieu in a highly efficient manner means that a vast number ofintracellular targets becomes druggable. Thus, a therapeutic protein ofinterest (Pol) is typically either a protein that binds to anintracellular target (for instance an intrabody against an oncogenicprotein such as c-Myc or a decoy receptor binding its intracellularinteraction partner) or a Pol that is meant to exert a desired effectintracellularly (the Pol may for instance be dystrophin as a treatmentof Duchenne's muscular dystrophy (DMD), a Pol for replacement of amissing or defect protein (such an enzyme like NPC1, GBA, or AGAL, etc.for enzyme replacement therapy, the Huntingtin protein or BDNF for thetreatment of e.g. Huntington's disease or other neurodegenerativedisorders), a tumor suppressor such as p53 for treatment of cancer, oran NFkB inhibitor for treatment of inflammatory diseases. Targets ofinterest for intrabodies delivered with the aid of the EVs of thepresent invention may include pathological forms of alpha-synuclein,LRRK2, Tau, Beta amyloid, APP, C9orf72, SOD1, TDP43, FUS and prionproteins. One class of Pols with considerable therapeutic potential arethe RNA-binding proteins (RBPs), which may be used to aid intracellulardelivery of RNA therapeutics such as mRNA, RNAi agents such asshort-hairpin RNA or microRNA, or antisense agents for splice-switchingor silencing. Non-limiting examples of RNA-binding proteins are hnRNPA1,hnRNPA2B1, DDX4, ADAD1, DAZL, ELAVL4, IGF2BP3, SAMD4A, TDP43, FUS, FMR1,FXR1, FXR2, EIF4A1-3, the MS2 coat protein, as well as any domains,parts or derivates, thereof. More broadly, particular subclasses ofRNA-binding proteins and domains, e.g. mRNA binding proteins (mRBPs),pre-rRNA-binding proteins, tRNA-binding proteins, small nuclear ornucleolar RNA-binding proteins, non-coding RNA-binding proteins, andtranscription factors (TFs). Furthermore, various domains andderivatives may also be used as the Pol for transport of an RNA cargo.Non-limiting examples of RNA-binding Pol include small RNA-bindingdomains (RBDs) (which can be both single-stranded and double-strandedRBDs (ssRBDs and dsRBDs) such as DEAD, KH, GTP_EFTU, dsrm, G-patch,IBN_N, SAP, TUDOR, RnaseA, MMR-HSR1, KOW, RnaseT, MIF4G, zf-RanBP, NTF2,PAZ, RBM1CTR, PAM2, Xpo1, Piwi, CSD, and Ribosomal_L7Ae. SuchRNA-binding domains may be present in a plurality, alone or incombination with others, and may also form part of a larger RNA-bindingprotein construct as such, as long as their key function (i.e. theability to transport an RNA cargo of interest, e.g. an mRNA or a shortRNA) is maintained.

Further as mentioned above, the exosomal polypeptides as per the presentinvention may be essentially any suitable polypeptide that enablestransport of the at least one Pol into an EV. As above-mentioned, theactual localization of the Pol after it has been transported into the EVmay vary depending on the nature of the exosomal polypeptide and/or thenature of the Pol, i.e. the Pol may be transported into the lumen of theEV, into the EV membrane, to a membrane-associated location, and/or toany other suitable part of the EV. Non-limiting examples of suchexosomal polypeptides are for instance CD81, Itab1, Mfge8, CD63, CD151,Hspg2, Lgals3 bp, Col6a1, Agrn, Tspan14, Lamc1, Lamb1, Tfrc, CD47, CD82,Slit2, Syntenin, Alix, Syndecan, synaptotagmin, Lamp2, Lamp2b, CD13,CD86, Flotillin, Syntaxin-3, LiCAM, LFA-1, Mac-1 alpha and beta, Vti-1Aand B, ICAM-1, CD2, CD18, CD37, CD36, CD53, CD82, CXCR4, FcR, CD40,CD40L, CD41a, CD44, CD45, and tetraspanins, GluR2/3, HLA-DM,immunoglobulins, MHC-I or MHC-II and components thereof, and TCR beta,and numerous other polypeptides capable of transporting a polypeptideconstruct comprising a Pol to an EV.

In yet another embodiment, the EVs of the present invention furthercomprise at least one targeting moiety. Typically, the targeting moietyis present on the surface of the EV (i.e. protruding from the EVmembrane into the extravesicular environment), typically in the form offusion proteins between the targeting moiety and an EV protein, in orderto facilitate reaching the correct tissue or cell type in vivo and/or invitro. The EVs may also further comprise penetration enhancers, toincrease the penetration into selected tissues or compartments. Suchpenetration enhancers may be peptides or polypeptides expressed on thesurface of the EVs as fusion constructs with a suitable exosomalpolypeptide. The penetration enhancers may for instance becell-penetrating peptides (CPPs) (such as Tat, transportan, transportan10, poly-Arg, MPG, Pep-1, penetratin, etc.), antibodies (which maytarget cell surface receptors that facilitate internalization, e.g. thetransferrin receptor or the insulin receptor), or affibodies, or anyother type molecule that would increase the internalization and/ortissue penetration of the EVs. In analogy with the targeting moieties,the penetration enhancers may be expressed on the surface of the EVsthrough creation of fusion constructs between an exosomal polypeptideand at least one penetration enhancer.

In yet another aspect, the present invention pertains to a highlyeffective method for producing EVs with strong therapeutic efficacy inlarge quantities. The methods as per the present invention comprise thesteps of (a) introducing into a source cell at least one polynucleotideconstruct which encodes at least one Pol, an endogenously activatablepolypeptide-based release system, and an exosomal polypeptide, and (b)expressing the corresponding polypeptide(s) from the polynucleotideconstruct(s). Typically, the method also comprises a step (c) ofcollecting the EVs generated (i.e. released) by the source cell, intowhich EVs the Pol has been released through endogenous triggering of theprotein-based release domain.

The introduction of suitable polynucleotide constructs into a sourcecell (typically a cell culture comprising a suitable EV-producing celltype for production of EVs) may be achieved using a variety ofconventional techniques, such as transfection, virus-mediatedtransformation, electroporation, etc. Transfection may be carried outusing conventional transfection reagents such as liposomes, CPPs,cationic lipids or polymers, calcium phosphates, dendrimers, etc.Virus-mediated transfection is also a highly suitable methodology, andmay be carried out using conventional virus vectors such as adenoviralor lentiviral vectors. Virus mediated transformation is particularlyrelevant when creating stable cell lines for cell banking, i.e. thecreation of master cell banks (MCBs) and working cell banks (WCBs) ofEV-producing cell sources.

In certain instances, it may be advantageous to introduce more than onepolynucleotide construct into the source cells. This may for instance bethe case when employing the NLS-NLSBP release system. In such cases, thepolypeptide constructs encoded for by the polynucleotide constructs willbe translated separately followed by the desired specific interactionbetween the polypeptides (e.g. a Pol comprising an NLS and an exosomalprotein fused to an NLSBP) inside the source cell post translation. Ifone is on the other hand employing a monomeric system such as aDendra-based release system or a cis-cleaving release system (e.g. anintein release system) it may be more advantageous to introduce a singlepolynucleotide construct into the source cell in order to encode for asingle polypeptide construct. In a further embodiment, the production ofEVs by the cells of the cell culture may be enhanced by exposing thecells to different conditions that may induce increased EV production.Serum starvation, hypoxia, exposure to cytokines such as TNFalpha orinterferons, antibiotics such bafilomycin, and other substances aremethods that may be used to increase the EV production, the yield, andalso the quality of the EVs.

In further aspects, the present invention also pertains to inventivepolynucleotide and polypeptide constructs. The polynucleotide constructsas per the present invention typically comprise nucleotide stretchesencoding for at least one Pol, at least one endogenously activatablepolypeptide-based release system or a portion of a polypeptide-basedrelease system, and at least one exosomal polypeptide. A non-limitingexample would be a polynucleotide construct encoding for an enzyme Pol(such as NPC1 or GBA) for the treatment of a lysosomal storagedisorders, a cis-cleaving intein, and an exosomal polypeptide such asCD81, syntenin or CD63. Thus, the present invention naturally alsorelates to the corresponding polypeptide constructs, i.e. polypeptideconstructs comprising at least one Pol, at least one endogenouslytriggered polypeptide-based release system or a portion of apolypeptide-based release system, and at least one exosomal polypeptide.Furthermore, the present invention also pertains to EV-producing cells(typically cells present in the form of cell culture but also individualcells as such) comprising the above-mentioned polynucleotideconstruct(s) and/or the above-mentioned polypeptide(s).

In another aspect, the present invention relates to methods for deliveryof a Pol into the intracellular environment or into the membrane of atarget cell, either in vitro or in vivo. The methods comprise contactinga target cell with an EV comprising either (i) a Pol which is releasablyattached to an exosomal polypeptide using an endogenously activatablepeptide-based release system or (ii) a Pol which has been released froman exosomal polypeptide, either inside the EV or inside the target cell.Importantly, unlike in the prior art, the Pols of the present inventionare delivered into target cells in a substantially unconjugated form,i.e. a Pol in question is not conjugated to a large exosomal protein (orin the case of a dimeric optogenetic proteins, an optogenetic dimer)that could potentially hamper the activity of the Pol. For instance,WO2014/168548 teaches exosomes comprising therapeutic polypeptides ofinterest that are conjugated to exosomal proteins, which is an excellentstrategy for certain types of therapeutic proteins that are capable ofexerting their intended activity despite being conjugated to an exosomalprotein. However, the methods of the present invention enable deliveryof a much wider range of therapeutic Pols, through the inventivecontrollable endogenously activatable release systems which liberate thePols in their desired location(s) without the need for any exogenousstimuli.

In certain embodiments as per the present invention, the Pol may be anintegral membrane protein conjugated to an exosomal polypeptide with theaid of the endogenously triggered protein-based release systems. Anintegral membrane Pol may be presented on the outer, inner or bothsurfaces of an EV. Without wishing to be bound by any theory, it issurmised that following uptake into a target cell, an EV—comprising apolypeptide construct which in turn comprises an integral membranePol—is trafficked to the endoplasmic reticulum (ER). The contents of theEV, i.e. the polypeptide construct comprising the integral membrane Pol,may be processed and sorted at the ER followed by ER-mediatedtrafficking to the appropriate compartment of the target cell. Thus, apolypeptide construct comprising an exosomal protein conjugated with theaid of a polypeptide-based release system to an integral plasma membraneprotein (for instance a G-protein coupled receptor (GPCR)) would berouted to the plasma membrane of a target cell, whereas a membraneprotein natively present in a lysosomal membrane (i.e. a lysosomalmembrane protein) would be routed to a lysosome of the target cell. Oneparticularly important example of a membrane Pol is NPC1, which is amembrane transporter of cholesterol and which when defect results in thestorage disorder Niemann-Pick's disease.

The release of the Pol is as above-outlined mediated by an endogenouslyactivatable polypeptide-based release system which is fused to the Poland/or to the exosomal polypeptide. In an advantageous embodiment, thepolynucleotide construct encoding for the subsequent polypeptideconstruct is designed in such a way so as to place the release system inbetween the Pol and the exosomal polypeptide. This arrangement enableseasy manufacturing of the constructs and efficient release of the Pol inthe desired location.

In yet another aspect, the present invention pertains to pharmaceuticalcompositions comprising EVs in accordance with the present invention.Typically, the pharmaceutical compositions as per the present inventioncomprise at least one type of therapeutic EV (i.e. a population of EVshaving comprising a certain desired Pol) formulated with at least onepharmaceutically acceptable excipient. The at least one pharmaceuticallyacceptable excipient may be selected from the group comprising anypharmaceutically acceptable material, composition or vehicle, forinstance a solid or liquid filler, a diluent, an excipient, a carrier, asolvent or an encapsulating material, which may be involved in e.g.suspending, maintaining the activity of or carrying or transporting thetherapeutic delivery vesicles from one organ, or portion of the body, toanother organ, or portion of the body (e.g. from the blood to any tissueand/or organ and/or body part of interest).

The present invention also relates to cosmetic applications of the EVscomprising Pols. Thus, the present invention pertains to skin careproducts such as creams, lotions, gels, emulsions, ointments, pastes,powders, liniments, sunscreens, shampoos, etc., comprising a suitableEV, in order to improve and/or alleviate symptoms and problems such asdry skin, wrinkles, folds, ridges, and/or skin creases. In oneembodiment of both cosmetic and therapeutic nature, the EVs as per thepresent invention may comprise a botulinum toxin (e.g. botox, forinstance botulinum toxin types A-G) as the Pol (botulinum toxins may notnecessarily be used only for cosmetic applications but could also beapplied for e.g. treatment of migraine headaches and dystonia). In apreferred embodiment, EVs (which comprise a at least one type of Pol)obtainable from a suitable exosome-producing cell with regenerativeproperties (such as a mesenchymal stem cell) are comprised in a cosmeticcream, lotion, or gel for use in the cosmetic or therapeutic alleviationof wrinkles, lines, folds, ridges and/or skin creases.

In yet another aspect, the present invention relates to EVs as per thepresent invention for use in medicine. Naturally, when an EV comprisinga Pol in accordance with the present invention is used in medicine, itis in fact normally a population of EVs that is being used. The dose ofEVs administered to a patient will depend on the amount of Pol that hasbeen loaded into the EV, the disease or the symptoms to be treated oralleviated, the administration route, the pharmacological action of thePol itself, as well as various other parameters of relevance.

The EVs of the present invention may be used for prophylactic and/ortherapeutic purposes, e.g. for use in the prophylaxis and/or treatmentand/or alleviation of various diseases and disorders. A non-limitingsample of diseases wherein the EVs as per the present invention may beapplied comprises Crohn's disease, ulcerative colitis, ankylosingspondylitis, rheumatoid arthritis, multiple sclerosis, systemic lupuserythematosus, sarcoidosis, idiopathic pulmonary fibrosis, psoriasis,tumor necrosis factor (TNF) receptor-associated periodic syndrome(TRAPS), deficiency of the interleukin-1 receptor antagonist (DIRA),endometriosis, autoimmune hepatitis, scleroderma, myositis, stroke,acute spinal cord injury, vasculitis, Guillain-Barré syndrome, acutemyocardial infarction, ARDS, sepsis, meningitis, encephalitis, liverfailure, kidney failure, graft-vs-host disease, Duchenne's musculardystrophy and other muscular dystrophies, lysosomal storage diseasessuch as Gaucher disease, Fabry's disease, MPS I, II (Hunter syndrome),and III, Niemann-Pick disease, Pompe disease, etc., neurodegenerativediseases including Alzheimer's disease, Parkinson's disease,Huntington's disease and other trinucleotide repeat-related diseases,dementia, ALS, cancer-induced cachexia, anorexia, diabetes mellitus type2, and various cancers.

Virtually all types of cancer are relevant targets for the presentinvention, for instance, Acute lymphoblastic leukemia (ALL), Acutemyeloid leukemia, Adrenocortical carcinoma, AIDS-related cancers,AIDS-related lymphoma, Anal cancer, Appendix cancer, Astrocytoma,cerebellar or cerebral, Basal-cell carcinoma, Bile duct cancer, Bladdercancer, Bone tumor, Brainstem glioma, Brain cancer, Brain tumor(cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodermaltumors, visual pathway and hypothalamic glioma), Breast cancer,Bronchial adenomas/carcinoids, Burkitt's lymphoma, Carcinoid tumor(childhood, gastrointestinal), Carcinoma of unknown primary, Centralnervous system lymphoma, Cerebellar astrocytoma/Malignant glioma,Cervical cancer, Chronic lymphocytic leukemia, Chronic myelogenousleukemia, Chronic myeloproliferative disorders, Colon Cancer, CutaneousT-cell lymphoma, Desmoplastic small round cell tumor, Endometrialcancer, Ependymoma, Esophageal cancer, Extracranial germ cell tumor,Extragonadal Germ cell tumor, Extrahepatic bile duct cancer, Eye Cancer(Intraocular melanoma, Retinoblastoma), Gallbladder cancer, Gastric(Stomach) cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinalstromal tumor (GIST), Germ cell tumor (extracranial, extragonadal, orovarian), Gestational trophoblastic tumor, Glioma (glioma of the brainstem, Cerebral Astrocytoma, Visual Pathway and Hypothalamic glioma),Gastric carcinoid, Hairy cell leukemia, Head and neck cancer, Heartcancer, Hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngealcancer, Intraocular Melanoma, Islet Cell Carcinoma (Endocrine Pancreas),Kaposi sarcoma, Kidney cancer (renal cell cancer), Laryngeal Cancer,Leukemias ((acute lymphoblastic (also called acute lymphocyticleukemia), acute myeloid (also called acute myelogenous leukemia),chronic lymphocytic (also called chronic lymphocytic leukemia), chronicmyelogenous (also called chronic myeloid leukemia), hairy cellleukemia)), Lip and Oral, Cavity Cancer, Liposarcoma, Liver Cancer(Primary), Lung Cancer (Non-Small Cell, Small Cell), Lymphomas((AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-Cell lymphoma,Hodgkin lymphoma, Non-Hodgkin (an old classification of all lymphomasexcept Hodgkin's) lymphoma, Primary Central Nervous System lymphoma)),Medulloblastoma, Merkel Cell Carcinoma, Mesothelioma, MetastaticSquamous Neck Cancer with Occult Primary, Mouth Cancer, MultipleEndocrine Neoplasia Syndrome, Multiple Myeloma/Plasma Cell Neoplasm,Mycosis Fungoides, Myelodysplastic/Myeloproliferative Diseases,Myelogenous Leukemia, Chronic Myeloid Leukemia (Acute, Chronic),Myeloma, Nasal cavity and paranasal sinus cancer, Nasopharyngealcarcinoma, Neuroblastoma, Oral Cancer, Oropharyngeal cancer,Osteosarcoma/malignant fibrous histiocytoma of bone, Ovarian cancer,Ovarian epithelial cancer (Surface epithelial-stromal tumor), Ovariangerm cell tumor, Ovarian low malignant potential tumor, Pancreaticcancer, Pancreatic islet cell cancer, Parathyroid cancer, Penile cancer,Pharyngeal cancer, Pheochromocytoma, Pineal astrocytoma, Pinealgerminoma, Pineoblastoma and supratentorial primitive neuroectodermaltumors, Pituitary adenoma, Pleuropulmonary blastoma, Prostate cancer,Rectal cancer, Renal cell carcinoma (kidney cancer), Retinoblastoma,Rhabdomyosarcoma, Salivary gland cancer, Sarcoma (Ewing family of tumorssarcoma, Kaposi sarcoma, soft tissue sarcoma, uterine sarcoma), Sezarysyndrome, Skin cancer (nonmelanoma, melanoma), Small intestine cancer,Squamous cell, Squamous neck cancer, Stomach cancer, Supratentorialprimitive neuroectodermal tumor, Testicular cancer, Throat cancer,Thymoma and Thymic carcinoma, Thyroid cancer, Transitional cell cancerof the renal pelvis and ureter, Urethral cancer, Uterine cancer, Uterinesarcoma, Vaginal cancer, Vulvar cancer, Waldenström macroglobulinemia,and/or Wilm's tumor.

The EVs as per the present invention may be administered to a human oranimal subject via various different administration routes, for instanceauricular (otic), buccal, conjunctival, cutaneous, dental,electro-osmosis, endocervical, endosinusial, endotracheal, enteral,epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration,interstitial, intra-abdominal, intra-amniotic, intra-arterial,intra-articular, intrabiliary, intrabronchial, intrabursal,intracardiac, intracartilaginous, intracaudal, intracavernous,intracavitary, intracerebral, intracisternal, intracorneal, intracoronal(dental), intracoronary, intracorporus cavernosum, intradermal,intradiscal, intraductal, intraduodenal, intradural, intraepidermal,intraesophageal, intragastric, intragingival, intraileal, intralesional,intraluminal, intralymphatic, intramedullary, intrameningeal,intramuscular, intraocular, intraovarian, intrapericardial,intraperitoneal, intrapleural, intraprostatic, intrapulmonary,intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular,intrathecal, intrathoracic, intratubular, intratumor, intratym panic,intrauterine, intravascular, intravenous, intravenous bolus, intravenousdrip, intraventricular, intravesical, intravitreal, iontophoresis,irrigation, laryngeal, nasal, nasogastric, occlusive dressing technique,ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous,periarticular, peridural, perineural, periodontal, rectal, respiratory(inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival,subcutaneous, sublingual, submucosal, topical, transdermal,transmucosal, transplacental, transtracheal, transtympanic, ureteral,urethral, and/or vaginal administration, and/or any combination of theabove administration routes.

In a further aspect, the present invention relates to, asabove-mentioned, a method of producing EVs (or more accurately producingpopulations of EVs) comprising the steps of (a) introducing into a cellsource (typically a cell culture) one or more polynucleotideconstruct(s) encoding at least one Pol, an endogenously activatablepolypeptide-based release system, and an exosomal polypeptide, (b)expressing the polypeptide construct(s) encoded by the polynucleotideconstruct(s), and (c) collecting EVs generated by the cell. If oneutilizes the monomeric light-induced cleave system for releasing the Polthen an additional step of short-term exposing the EVs to light of asuitable wavelength is added to the production method. The lightexposure step can take place while the EVs are still being formed insidecells, when the EVs have just been released into the cell culturemedium, when EVs have been processed further (for instance by tangentialflow filtration (TFF), ultrafiltration, bead-elute chromatography,size-exclusion chromatography or any combination thereof), oressentially whenever suitable depending on the cell source, thecharacteristics of the EV production, and the EVs per se. Suitableculture systems include conventional 2D cell culture, 3D cell culture,bioreactors, hollow-fiber bioreactors, etc.

The method may also comprise exposing the source cells to serumstarvation, hypoxia, bafilomycin, or cytokines such as TNF-alpha and/orIFN-gamma, in order to influence the yield or properties of theresulting EVs. The EV production scale and timeline will be heavilydependent on the EV-producing cell or cell line and may thus be adaptedaccordingly by a person skilled in the art.

The production methods may further comprise a purification step, whereinthe EVs are purified through a procedure selected from the groupcomprising liquid chromatography (LC), bead-elute LC, size-exclusion LC,high-performance liquid chromatography (HPLC), spin filtration,tangential flow filtration, hollow fiber filtration, centrifugation,immunoprecipitation, etc, or any combination thereof.

In an advantageous embodiment, the purification of the EVs is carriedout using a sequential combination of filtration (preferablyultrafiltration (UF), tangential flow filtration (TFF) or hollow fiberfiltration) and bead-elute or size exclusion liquid chromatography (LC).This combination of purification steps results in optimizedpurification, which in turn leads to superior therapeutic activity.Further, as compared to ultracentrifugation (UC), which is routinelyemployed for purifying exosomes, sequential filtration-chromatography isconsiderably faster and possible to scale to higher manufacturingvolumes, which is a significant drawback of the current UC methodologythat dominates the prior art.

It shall be understood that the above described exemplifying aspects,embodiments, alternatives, and variants can be modified withoutdeparting from the scope of the invention. The invention will now befurther exemplified with the enclosed examples, which naturally also canbe modified considerably without departing from the scope and the gistof the invention.

Experimental Part Materials and Methods

Construct Design and Cloning

Various cis-cleaving endogenously activatable polypeptide-based releasesystems (such as slow-cleaving inteins, fast-cleaving inteins, sortase Aand FrpC) have been assessed, in combination with several Pols (such asCre, NPC1, IL10, RNA-binding MS2 coat protein) and exosomal polypeptides(CD81, CD63, CD9, syntenin, syndecan, Alix, CD133, etc.). Similarly,various NLS-NLSBP release systems and monomeric light induced-releasesystems were assessed, together with Pols and EV proteins. NLS-NLSBPsystems include: KPNA1-NRF2, KPNA6-STAT3, KPNB1-STAT3, KPNA2-HSF1.NLSBPs include importins from the importin alpha and beta families, andother NLS binding proteins, including KPNA1, KPNA2, IPO8, TPNO1,HIKESHI, SNUPN, HEATR3, and other RAN binding proteins. NLS containingproteins include various transcription factors, nucleases and othernuclear proteins such as bHLH, MyoD, cMyc, HSF1, STAT3, p53, NFkB, Cas9,HSP70, and U1 snRNP. Monomeric light-induced cleavage systems include:Kaede, KikGR, EosFP, and Dendra. Pols include NPC1, GBA, AGAL,Huntingtin, BDNF, an NFkB super-repressor, RNA-binding proteins anddomains such as hnRNPA1, the MS2 coat protein, G-patch, etc. In the caseof RNA-binding proteins, these have been combined with a polynucleotideof interest, which said RNA-binding proteins drag into the EV. A largenumber of exosomal proteins have been evaluated: CD81, CD63, CD9,syndecan, Alix, CD133, Syntenin-1, Syntenin-2, Lamp2b, TSPAN8, andTSPAN14, as well as variants and domains thereof.

When evaluating the cis-cleaving release system constructs and themonomeric light-induced cleavage systems, ORFs were typically generatedby synthesis and cloned into the mammalian expression vectorpSF-CAG-Amp. Briefly, synthesized DNA and vector plasmid were digestedwith enzymes NotI and SalI as per manufacturers instruction (NEB).Restricted, purified DNA fragments were ligated together using T4 ligaseas per manufacturers instruction (NEB). Successful ligation events wereselected for by bacterial transformation on ampicillin-supplementedplates. Plasmid for transfection was generated by ‘maxi-prep’, as permanufacturers instruction.

In the case of the NLS-NLSBP release system, ORF sequences were normallypurchased (Origene Technologies, Inc.) and amplified and cloned into theMSC A site of pIRES bicistronic expression vector (Clonetech,Laboratories Inc.) such that upon translation the NLSBP protein wasexpressed as a chimera, fused to the exosomal polypeptide. NLS wereeither fused onto Pols lacking NLS, or when already present on the Polused as-is, occasionally with genetic modifications. Most of the cloningwas performed using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB,Inc.) and confirmed using Sanger sequencing (Source BioScience). ThepIRES vector enables bicistronic expression of both transgenessimultaneously, ensuring EV-producing cells would express bothtransgenes simultaneously. Plasmids were transformed into the NEB5-alpha Competent E. coli cells (NEB, Inc.) and grown overnight in ashaking incubator according to manufacturer's recommendations. Plasmidswere isolated and purified using the ‘maxi-prep’ kit, as permanufacturer's instruction (Macherey-Nagel).

Cell Culture and Transfection

Depending on the experimental design and assays, in certain cases,non-viral transient transfection and exosome production was carried outin conventional 2D cell culture, whereas in other cases virus-mediatedtransduction was employed to create stable cell lines, which weretypically cultured in bioreactors of different type. For conciseness,only a few examples are mentioned herein.

HEK293T cells were typically seeded into 15 cm dishes (9×10⁶ cells perdish) and left overnight in serum-containing DMEM as recommended byATCC. The following day the cells were transiently transfected withlipoplexed DNA added directly onto cells. Briefly, DNA andpolyethyleneimine (PEI) were separately incubated in OptiMEM for 5minutes before combining together for 20 minutes at room temperature.Lipoplexed DNA and cells were co-incubated for 6 hours following whichconditioned culture media was changed to OptiMEM for 48 hours. Othercells and cell lines that were evaluated in dishes, flasks and othercell culture vessels included bone marrow-derived mesenchymal stromalcells (BM-MSCs) and Wharton's jelly-derived MSCs (WJ-MSCs), amnioncells, fibroblasts, various endothelial and epithelial cells, as well asvarious immune cells and cell lines.

In the case of viral transduction and creation of stable cell lines forvarious combinations of Pol, release system, and exosomal polypeptide,cell sources such as BM-MSCs, WJ-MSC, fibroblasts, amnion cells,fibroblasts, various endothelial and epithelial cells, werevirus-transduced, typically using lentivirus (LV). Typically, 24 hoursbefore infection, 100.000 cells (e.g. fibroblasts, MSCs, etc.) or200.000 cells (e.g. HEK293T) are plated in a 6-well plate. 2 uL of LVand optionally Polybrene (or hexadimethrine bromide, final concentrationon the well of 8 ug/mL) are added, and 24 hours post transduction thecell medium of transduced cells is changed to fresh complete media. At72 hours post transduction, puromycin selection (4-6 μg/ml) isperformed, normally for 7 days followed by analysis of stable expressionof the polypeptide construct.

Stable cells were cultured in either 2D culture or in bioreactors,typically hollow-fiber bioreactors, and conditioned media wassubsequently harvested for exosome preparation. Various preparation andpurification steps were carried out. The standard workflow comprises thesteps of pre-clearing of the supernatant, filtration-basedconcentration, chromatography-based removal of protein contaminants, andoptional formulation of the resultant exosome composition in a suitablebuffer for in vitro and/or in vivo assays.

Assays and Analytics

Western blot is a highly convenient analytical method to evaluate theenrichment of Pols in EVs. Briefly, SDS-PAGE was performed according tomanufacturer's instruction (Invitrogen, Novex PAGE 4-12% gels), whereby1×10¹⁰ exosomes and 20 ug cell lysate were loaded per well. Proteinsfrom the SDS-PAGE gel were transferred to PVDF membrane according tomanufacturer's instruction (Immobilon, Invitrogen). Membranes wereblocked in Odyssey blocking buffer (Licor) and probed with antibodiesagainst Pol and the exosomal protein according to supplier's instruction(Primary antibodies—Abcam, Secondary antibodies—Licor). Molecular probesvisualized at 680 and 800 nm wavelengths. FIG. 14 shows an illustrationof this for the combination of Cre recombinase as the Pol and Alix asthe exosomal protein.

For EV size determination, nanoparticle tracking analysis (NTA) wasperformed with a NanoSight instrument equipped with analytical software.For all recordings, we used a camera level of 13 or 15 and automaticfunction for all post-acquisition settings. Electron microscopy andfluorescence microscopy were frequently used to understand Pol locationand release and to quantitate and analyze EVs.

EVs were isolated and purified using a variety of methods, typically acombination of filtration such as TFF and LC. Typically, EV-containingmedia was collected and subjected to a low speed spin at 300 g for 5minutes, followed by 2000 g spin for 10 minutes to remove largerparticles and cell debris. The supernatant was then filtered with a 0.22μm syringe filter and subjected to different purification steps. Largevolumes were diafiltrated and concentrated to roughly 20 ml using theVivaflow 50R tangential flow (TFF) device (Sartorius) with 100 kDacutoff filters or the KR2i TFF system (SpectrumLabs) with 100 or 300 kDacutoff hollow fibre filters. The preconcentrated medium was subsequentlyloaded onto the bead-eluate columns (HiScreen or HiTrap Capto Core 700column, GE Healthcare Life Sciences), connected to an AKTAprime plus orAKTA Pure 25 chromatography system (GE Healthcare Life Sciences). Flowrate settings for column equilibration, sample loading and columncleaning in place procedure were chosen according to the manufacturer'sinstructions. The sample was collected according to the UV absorbancechromatogram and concentrated using an Amicon Ultra-15 10 kDa molecularweight cut-off spin-filter (Millipore) to a final volume of 100 μl andstored at −80° C. for further downstream analysis. To assess the proteinand RNA elution profiles, media was concentrated and diafiltrated withKR2i TFF system using 100 kDa and 300 kDa hollow fibre filters and asample analysed on a Tricorn 10/300 Sepharose 4 Fast Flow (S4FF) column(GE Healthcare Life Sciences).

Examples

FIG. 3 shows the results of GBA-deficient patient-derived lymphocytestreated with BM-MSC EVs enriched with a polypeptide construct comprisinga slow-cleaving intein inserted between the GBA enzyme and the exosomalprotein CD63. Using an H PLC-based assay, recipient cells were shown todisplay a decrease in the levels of the lipid glucocerebroside followingthe EV-mediated delivery of freely released GBA. A similar experimentwas carried out using a polypeptide construct comprising as Pol thelipid transporter protein NPC1, a fast-cleaving intein-based releasesystem, and the exosomal protein CD133. EVs comprising this polypeptideconstruct efficiently delivered NPC1 to NPC1-deficient fibroblasts,leading to a significantly higher level of functional cholesteroltransport as opposed to control EVs comprising a non-functional releasesystem.

In another example, an endogenously activatable N-terminusprotease-based release system was used to fuse the Pol p53 to theexosomal protein syndecan, with fibroblasts as the EV-producing parentalcell. EV-mediated endogenously actived releasable delivery of p53 intoMDA-MB-231 cancer cells in vitro was significantly more effective thannon-releasable delivery using the same EV polypeptide construct.

FIG. 5 shows the results of a non-homologous end-joining (NHEJ) assay.HEK293T-red cells containing a reporter system were transfected withfibroblast-derived EVs comprising guide RNA (gRNA) and a polypeptideconstruct comprising CD81 as the exosomal polypeptide, Kaede as themonomeric light-induced cleavage-based release system, and Cas9 as thePol. EVs where obtained from a cell culture that either was or was notexposed to blue light during the exosome production process. Onlyexosomes obtained from cells exposed to a short boost of light—whichinduced cleavage of Kaede and thereby release of Cas9—showed an increasein the percentage of positive cells. Similarly, FIG. 6 shows the resultsof a High Resolution Melting (HRM) analysis of cells treated with EVsharvested from cells exposed to light. Fibroblast EVs comprising thesame polypeptide construct as in FIG. 5 induced Cas9-mediated mutationsin the AAVS locus as a result of efficient intracellular delivery ofbioactive Cas9 and gRNA. FIG. 7 shows the effects in the NHEJ assay ofamnion epithelial cell-derived EVs comprising a polypeptide constructcomprising Cas9 fused to CD63 through the monomeric light-inducedrelease polypeptide Dendra2, which renders functional Cas9 in EVs afterUV/blue light irradiation. FIG. 8 shows HRM results of cells treatedwith WJ-MSC EVs harvested from cells exposed to UV/blue light. EVscomprising the same polypeptide construct (but this time inWJ-MSC-derived EVs) as in FIG. 7 induced Cas9-mediated mutations in theAAVS locus as a result of efficient intracellular delivery of bioactiveCas9 and gRNA. FIG. 9 shows robust down regulation of NfKB responseachieved by HEK EVs loaded with a WASP-targeted single-chain variablefragment (scFv)-KikGR-CD63 polypeptide construct, exposed to UV-light torelease the scFv inside the target bone marrow— derived macrophage cell.

FIG. 10 shows the results of Jurkat cells stimulated to express theIL2-receptor and at the same time treated with HEK EVs loaded with ascFv towards the IL2R-alpha subunit. Only the positive control and theEVs loaded with scFv-Dendra2-CD63 (and exposed to UV-light) induced adown regulation of the IL2R on the cell surface of the Jurkat cells,according to FACS analysis. FIG. 11 shows the results of theerbB-2-positive ovarian carcinoma cell line SKOV3 treated with EVsloaded with a scFv targeted towards the oncoprotein erb-2. Cell deathwas assayed at 48 hours after treatment. Only EVs loaded withscFv-Dendra2-CD63 (and thereafter briefly exposed to UV-light) inducedcell death in comparable levels to the positive control.

FIG. 12 shows the results of loading NRF2 transcription factor toBM-MSC-derived EVs using the NLSBP-NLS-based release system and theassociated effects in inducing target gene HMOX1 expression in recipientcells. An NLSBP (KPNA1, also known as importin α5) was fused to theexosomal protein CD63 and co-expressed in EV source cells (in additionto BM-MSCs, various types of immune cells were tested with good results)with NRF2. Co-expression leads to significantly enhanced EV sorting ofNRF2, as estimated by Western blotting as compared to expression of NRF2alone. Delivery of NRF2 loaded-EVs using this strategy leads toinduction of target gene expression in a HEK cell assay. Generatingpolypeptide construct based on other EV proteins also resulted insimilar effect, for instance when using Alix and syntenin. FIG. 13 showsa similar experiment as in FIG. 5 , but here with a cis-cleaving intein(comprising the amino acid sequence Val-Val-Val-His-Asn (SEQ ID NO: 1)as a release system fused to CD63, CD81 (data not shown), and syntenin(data not shown) and to Cas9, and delivered using amino epithelialcells. As can be seen from FIG. 13 , only the unmutated intein inducedrelevant levels of NHEJ.

Another example of the NLS-NLSBP polypeptide-based endogenouslyactivated release system includes the fusion of KPNA2 to exosomalprotein CD47 and co-expressing it with the transcription factor HSF1 infibroblasts, thus loading HSF1 into exosomes. The fibroblast-derivedexosomes were isolated and used to treat mouse primary cerebellargranule neurons that had been subjected to low or high potassiumconcentration in their culture media. HSF1-loaded EV treatment led tosignificantly higher cell viability compared to the treatment withcontrol exosomes without the NLS-NLSBP release system atapoptosis-inducing low potassium conditions.

What is claimed is:
 1. A method for producing extracellular vesicles(EVs) comprising: (i) introducing into an EV-producing cell at least onepolynucleotide construct encoding at least one polypeptide constructcomprising at least one polypeptide of interest (Pol), an endogenouslyactivatable polypeptide-based release system, and at least one exosomalpolypeptide; (ii) expressing in the EV-producing cell at least onepolypeptide construct encoded for by the at least one polynucleotideconstruct; and (iii) collecting from the EV-producing cell Evscomprising a Pol.
 2. The method of claim 1, wherein thepolypeptide-based release system is a cis-cleaving release system or anuclear localization signal (NLS)-nuclear localization signal-bindingprotein (NLSBP) (NLS-NLSBP) release system.
 3. The method of claim 2,wherein the polypeptide-based release system is a cis-cleaving releasesystem; further wherein the cis-cleaving release system is an inteinrelease system.
 4. The method of claim 1, wherein the upon activation ofthe polypeptide-based release system, the at least one Pol is releasedfrom the at least one exosomal polypeptide into the lumen of the EV. 5.The method of claim 1, wherein the Pol is a therapeutic polypeptideselected from the group consisting of antibodies, intrabodies, singlechain variable fragments, affibodies, enzymes, tumor suppressors, viralor bacterial inhibitors, cell component proteins, DNA and/or RNA bindingproteins, DNA repair inhibitors, nucleases, proteinases, integrases,transcription factors, growth factors, apoptosis inhibitors andinducers, toxins, structural proteins, neurotrophic factors, membranetransporters, nucleotide binding proteins, heat shock proteins,CRISPR-associated proteins, and any combination thereof.
 6. The methodof claim 5, wherein the at least one polypeptide of interest is (i) aCRISPR-associated (Cas) polypeptide with intact nuclease activity or(ii) a catalytically inactive CRISPR-associated (Cas) polypeptide. 7.The method of claim 6, wherein the polypeptide of interest is acatalytically inactive CRISPR-associated (Cas) polypeptide capable ofenabling targeted genetic engineering.
 8. The method according to claim1, wherein the at least one exosomal polypeptide is a polypeptidecapable of transporting a polypeptide construct comprising the Pol to anEV.
 9. The method of claim 1, wherein the exosomal polypeptide isselected from the group consisting of CD9, CD53, CD63, CD81, CD54, CD50,FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, Syntenin-1,Syntenin-2, Lamp2b, TSPAN8, TSPAN14, CD37, CD82, CD151, CD231, CD102,NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d/ITGA4,ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18/ITGB2, CD41, CD49b,CD49c, CD49e, CD51, CD61, CD104, tetraspanins, Fc receptors, interleukinreceptors, immunoglobulins, MHC-I or MHC-II components, CD2, CD3epsilon, CD3 zeta, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L,CD44, CD45, CD45RA, CD47, CD86, CD110, CD111, CD115, CD117, CD125,CD135, CD184, CD200, CD279, CD273, CD274, CD362, COL6A1, AGRN, EGFR,GAPDH, GLUR2, GLUR3, HLA-DM, HSPG2, L1 CAM, LAMB1, LAMC1, LFA-1,LGALS3BP, Mac-1 alpha, Mac-1 beta, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD,TCRG, VTI1A, VTI1B, and other exosomal polypeptides.
 10. The method ofclaim 1, further comprising exposing the EV-producing cell to serumstarvation, hypoxia, bafilomycin, or one or more cytokine.
 11. Themethod of claim 10, wherein the one or more cytokine is TNF-alpha and/orIFN-gamma.
 12. The method of claim 1, further comprising step (iv)purifying the EVs.
 13. The method of claim 12, wherein the purifyingcomprises a procedure selected from the group consisting of liquidchromatography (LC), bead-elute LC, size-exclusion LC, high-performanceliquid chromatography (HPLC), spin filtration, tangential flowfiltration, hollow fiber filtration, centrifugation,immunoprecipitation.
 14. An EV produced by the method of claim 1.